The most effective existing method for controlling Scarabaeidae, such as the Japanese beetle, comprises infecting the larvae with host-specific bacteria that cause milky disease in these larvae. Milky disease is lethal to the larvae, but harmless to other species of animals or plants. Known milky disease bacteria include, but are not limited to, the various varieties of Bacillus popilliae (including the varieties popilliae, lentimorbus, melolonthae, rhopaea, N.Z. Type I, N.Z. Type II, etc.)
The vegetative (rod) stage of these bactieria is not suitable for use in insecticidal preparations. Rods are sensitive and do not survive under the conditions associated with insecticide application methods or under those prevailing in the fields. By contrast, the spores of these bacteria are very resistant to adverse environmental conditions and remain viable (and infective) in the field after application (in liquid, powder, granular, or bait formulations) and for prolonged period of time thereafter.
Because of the high mortality rate of milky disease, the host-specificity of the milky disease pathogens and the absence of the type of adverse environmental impact that usually accompanies use of chemical pesticides, the milky disease spores are particularly suitable for use in pesticidal compositions. However, various difficulties in obtaining effective spores, particularly in large and economically attractive quantities, have prevented such pesticides from gaining wide acceptance.
Many investigators have failed to obtain substantial sporulation of B. popilliae and other milky disease bacteria in vitro, Dutky first proposed a method for producing B. popilliae spores in vivo. This method, described, e.g., in U.S. Pat. No. 2,293,890, involves injecting live Japanese beetle larvae with viable B. popilliae spores (themselves obtained from the hemolymph of diseased larvae), waiting for the disease to develop, drying and powdering the diseased larvae, and applying the resulting material in the field.
It is evident that this in vivo method is extremely tedious, costly, and labor-intensive. Moreover, it can produce only limited amounts of B. popilliae product, both because the quantity of spores obtained as a percentage of the larvae mass is small, and because scarabaeid larvae can be obtained or grown only during certain months (March to May and August to October).
To satisfy the recognized need for an alternative source of milky disease spores, investigation turned to in vitro methods. Unfortunately, only limited sporulation of milky disease bacteria has been reported in vitro. Although large numbers of vegetative cells can be produced in artificial (liquid or solid) media, the average degree of sporulation does not usually exceed about 10-30% and the spore infectivity has been reported to be either substantially impaired (such that it would not be commercially useful) or nonexistent: see, M. G. Klein, "Advances in the Use of B. popilliae for Pest Control" in Micr. Contr. of Pests and Plant Diseases, Burgess, H.D. (Editor) 1981 Academic Press, pp. 184-192.
St. Julien and L. A. Bulla, Jr. Current Topics in Comparative Pathobiology, T. C. Cheng (Editor) 1973, G., Vol. 2, Academic Press, pp 57-87 report that as high as 20% sporulation occurs in a population of NRRL B-2309M colonial cells in solid medium formulated with yeast extract, the ingredients of Mueller-Hinton medium (1%), trehalose, and phosphate. Mueller-Hinton medium contains a very low amount (0.15%) of starch. Therefore, the St. Julien and Bulla medium contains only about 0.0015% of starch.
U.S. Pat. No. 3,308,038 of Rhodes et al is directed to a process for inducing in vitro sporulation of NRRLB-2309 substrains of B. popilliae to the extent of only 3-5% by (a) culturing vegetative cells for 18-24 hours in shaken flasks (or with 0.15-0.5 vvm aeration) in an aqueous medium containing (on a weight-per-volume basis) 0.2% glucose, fructose or trehalose, 1.5-2.0% yeast extract, and sufficient K.sub.2 HPO.sub.4 to adjust the pH to 7.2-7.5 (0.3%); (b) transferring the cells to slants or plates containing agar, yeast extract, sodium acetate, and K.sub.2 HPO.sub.4 to form uncrowded colonies; and (c) culturing the colonies for 42 days until sporulation occurs.
U.S. Pat. No. 3,071,519 of Bonnefoi is directed to a method for producing large number of spores of B. thuringiensis involving culturing a stock of this bacterium in a liquid medium at a pH between 5.5 and 8.5 containing aminated nitrogen and at least one of saccharase, maltose, dextrose and dextrin and, as a trace element, one or more of calcium, zinc, manganese and magnesium until sporulation occurs, and harvesting the spores. However, B. thuringiensis is not a milky disease bacterium. Moreover, B. thuringiensis has proved to be much easier to grow in vitro than the milky disease bacilli.
U.S. Pat. No. 3,503,851 to Srinivasan is directed to an in vitro method for producing B. popilliae spores comprising growing B. popilliae in a liquid medium containing yeast extract, glucose, glycerol, sodium chloride, ammonium sulfate, K.sub.2 HPO.sub.4, MnSO.sub.4.H.sub.2 O, CaCl.sub.2, ZnSO.sub.4.7H.sub.2 O, FeSO.sub.4.7H.sub.2 O and a small amount of a chloroaliphatic compound, such as a chloroacetamide, chloroform or a trichloroethane. The thud obtained sporulation rate is said to be as large as 80%.
First, the use of chloroaliphatic compounds is undesirable for two reasons: (a) these compounds are volatile and would need constant replenishment in an aerated culture medium, which adds to the production costs; and (b) these compounds are toxic--their vapors would present a hazard to plant personnel and residual amounts that would remain in the product, would be released into the environment and contaminate agricultural crops. For these reasons, Srinivasan's process is neither amenable to nor desirable for commercial-scale production.
Second, the strains deposited by Srinivasan are not B. popilliae. These strains are reported as having been misidentified in the Agricultural Handbook, infra, Table 50, pp 260-261 (citing Srinivasan's patent discussed above) and have been since identified as not B. popilliae (B. megaterium, B. cereus, B. polymyxa, etc.). The literature also contains other reports of B. cereus and B. polymyxa being confused with B. popilliae.
U.S. Pat. No. 3,950,225 of Skole is directed to a two-stage (fermentation-sporulation) method for B. popilliae and B. lentimorbus. Vegetative growth is accomplished in a medium containing yeast extract, K.sub.2 HPO.sub.4, glucose and triptose. The resulting cell mass is then transferred to and incubated in cane sugar refinery animal char waste water as a sporulation medium. The patent contends that a sporulation rate of 100% was obtained by this process but does not give the basis on which this figure was calculated. The patent is silent about spore counts on a per-unit-volume (of culture medium) basis. Furthermore, in the only example, the patent states that a cell mass of 6.5.+-.0.5 g was transferred from 75 ml of vegetative growth medium to 5 liters of waste char water. This represents a more than 66-fold increase in volume, which would be highly impractical on a commercial scale. Finally, the Skole patent contains no infectivity data on the in vitro-produced spores.
Thus, many investigators have published or patented detailed protocols for inducing sporulation of B. popilliae in vitro. However, none of these procedures have been found to meet the basic requirements of an efficient process for producing B. popilliae spores that would be suitable for large scale production.
These requirements are:
large cell populations should be produced per unit fermentation volume; PA0 a high percentage of the cells should sporulate; PA0 growth and sporulation should occur under conditions amenable to large scale processing; PA0 the resulting spores should be capable of germinating and should be infective. PA0 Apparently, in vitro spores of B. popilliae B-2309M are unable to infect Japanese beetle larvae through the natural pathway, the insect gut. [Sharpe et al, supra, at p. 686]
Currently, B. popilliae var. popilliae (which may contain small amounts of the lentimorbus variety) is the only milky disease bacillus that is produced commercially and it is produced only in vivo.
The failure of the prior art to provide an efficient process (especially one adaptable for commercial-size scale-up) for producing milky disease spores in vitro was accompanied (or possibly caused) by certain views about the morphology and properties of milky disease bacilli, and in particular, of B. popilliae and its varieties. These views are widely held by those skilled in the field as evidenced by numerous references in the literature and constitute the "accepted wisdom" in the field.
B. popilliae is universally characterized in the literature as an obligate insect pathogen formed in a swollen sporangium that do not autolyse to release a free spore (see. e.g., R. J. Milner, infra, p. 46). This bacterium is catalasenegative and unable to grow in nutrient broth. It is considered to have extremely fastidious requirements for growth.
The accepted view in the literature about B. popilliae spores is that they are almost always retained in a sporangium except in rare instances when the bacilli are grown on solid media in vitro and extremely rarely in vivo. Thus, the appearance of a sporangium is accepted as an important marker for identification of the species. In fact, well recognized investigators have stated that spores grown in liquid culture that doe not have a sporangium are not B. popilliae. See generally, the Agriculture Handbook, No. 427, R. E. Gordon, et al (Agricultural Research Service, U.S.D.A., 1973) and R. J. Milner, "Identification of the Bacillus popilliae Group of Insect Pathogens" in Micr. Contr. of Pests and Plant diseases, Burges, H. D. (Editor) 1981 Academic Press, pp. 45-59. Thus, even if investigators had observed naked (sporangium-free) milky disease spore in media other than those containing acetate, they would not have identified it as such. Sharpe, E. S. et al 1984 Appl. Microbiol. 19(4): 681-688 report the observation of occasional free spores in sporulating colonies (i.e. on solid media) of B. popilliae strain NRRL-B-2309M. The same investigators obtained marginal results in terms of in vitro rate of sporulation and--most significant--extremely poor infectivity on assay. The results of their per os infectivity tests (in which the soil was inoculated with 30.times.10.sup.6 spores per gram) were negative. These results prompted the investigators to state:
R. G. Milner, supra reports that the spore and parasporal body can be released from the sporangium by ultrasonics for the purpose of studying the details of the spore surface structure, but this does not involve naked spore directly harvested from liquid culture media.
Another proposition that is widely accepted in the field is that a bioassay, in which B. popilliae (or any milky disease spore) is administered to scarabaeid larvae by injection, provides a reliable test for the infectivity of the spores. This injection bioassay has been found to require a lower dose level for infection than those required in bioassays in which the spore is administered per os (such as the soil inoculation bioassay). Given that per os bioassays are substantially more difficult and time-consuming to perform, the injection assay was adopted as the infectivity indicator of choice. Moreover, spores found to be noninfective by injection have often been reported to be noninfective when administered per os. The converse has not been reported.
All of the observations mentioned above serve to construct in the minds of those skilled in the art the implicit conviction that is spores were not infective when administered by injection, they would not be infective when ad- ministered per os.
In summary, the state of the art (at the time the present invention was made) accepted the above-described statements on the morphology and properties of milky disease bacilli, and in particular of B. popilliae, and had not succeeded in efficiently growing B. popilliae in vitro on a large scale.